Time-lapse analysis of zygote formation in mating mixtures

GFP-Bud1 labeled MATα cells (XWY028) and experimental MATa cells were grown to mid-log phase in synthetic 2% dextrose medium, mixed at a 1:1 ratio, and spread at a density of 14,000 cells/mm² on agarose pads made from synthetic dextrose medium. Mating mixtures were maintained at 30°C with a DeltaVision environment control chamber (GE Healthcare Bio-Sciences). Images were acquired from 20 fields at 15-min intervals with a DeltaVision Elite microscope (GE Healthcare Bio-Sciences) with a 60× oil immersion objective and a Front Illuminated sCMOS digital camera. To follow details of cell growth and zygote formation, DIC images were acquired in 31 z-sections over 6 µm. To identify the MATα cells, fluorescent images (461 to 489 nm excitation) were acquired at the center slice. Images were processed with ImageJ software. Initial orientation angles, fusion angles, time of fusion, and morphologies of MATa cells were quantified and analyzed as described in Fig. 4.